When a cell is removed from its original tissue or organism and placed in culture, the medium must provide all the environmental conditions that the cell has been exposed to in vivo; only then will it be able to survive, to proliferate, and to differentiate or maintain its differentiated phenotype. Thus, extracellular medium must meet the essential requirements for survival and growth (i.e. must provide nutritional, hormonal, and stromal factors). Where cells are cultured as part of a system to produce biologicals, e.g., for the production of vaccines or recombinant protein, the culturing medium also should not interfere with the cell's production of the desired biological. Among the biological fluids that have proved successful for culturing cells outside the body, serum has gained the most widespread significance.
However, there are a number of disadvantages with serum-containing media, particularly as part of a recombinant biologicals production system, including difficulties with sterilization, inconsistent variation between serum batches, the presence of extraneous serum constituents including indefinable and potentially cytotoxic constituents from which the biological product of interest must be purified, and risk of contaminants. Accordingly, the art long has sought to create non-serum culture media (including "serum-free" and "chemically-defined" media) which provide the necessary environmental conditions for cell growth and viability. Defining components necessary for a non-serum culturing media has been an on-going effort in the art. In many non-serum culture media, cell growth often is slower, and cell density and saturation levels, as well as cell viability, may be diminished. In addition, media formulations found useful for cell growth often are not optimal for recombinant protein production, requiring a change in culture media when protein production is to be induced, or a compromise in desired protein production levels.
Mizrahi and Lazar (Cytotechnology (1988) 1:199-214) describes the general state of the art of serum-free, chemically defined media for ex vivo mammalian cell cultivation.
Along with an energy source, a nitrogen source, vitamins, inorganic salts, nucleic acid precursors, and oxygen, fats (lipids) and fat-soluble components are critical elements for non-serum containing culture medium. Among the lipids found to be useful in serum-free or chemically defined media are phospholipids commonly associated with the cell membrane, and various lipid and phospholipid precursors. Goodwin, et al (1990) Nature 347:209-210 describes lipid supplements useful in serum-free media. Bromke, et al (1986) J. Microbiol, Methods, 6:55-59 describe the use of cholestrol and oleic acid in serum-free media. Imagawa et al., (1989) PNAS 86:4122-4126 describe the use of the lipid precursors dilinoleoyl phosphatidic acid and phosphatidyl serine to stimulate growth of epithelial cells in a serum-free medium. Bashir et al. (1992) In Vitro Cell Dev. Biol. 28A:663-668 describes the use of the lipid precursors phosphatidic acid and lysophosphatidic acid to stimulate the growth of kidney cells in serum-free media.
International application PCT/US90/03430, published Apr. 15, 1990, discloses a serum-free cell culture medium for enhanced cell growth that includes the phospholipid precursors choline, ethanolamine, phosphatidyl choline and phosphatidyl ethanolamine. Miyazaki et al. (1991) Res. Exp. Med. 191:77-83 describe the use of phosphatidyl ethanolamine and phosphatidyl choline, as well as the phospholipid precursors ethanolamine and phosphoethanolamine, to prolong survival of rat hepatocytes in culture. Kovar (1987) Folia Biologica 33:377-384 describes the use of dipalmitoyl lecithin, cholesterol and linoleic acid to promote the growth of hybridomas in serum-free media.
It is an object of the invention to provide an improvement in serum-free cell culture media. Another object is to provide a cell culture media, and methods for its production, having improved cell cultivation properties, including enhanced cell growth and cell viability, and enhanced production of recombinant biologicals. Another object of this invention is to provide serum-free media having enhanced cell culture properties and which can be utilized in a wide range of cell culture systems for the recombinant production of biologicals, including proteins, using any of a number of cell lines and recombinant protein expression systems known in the art. Still another object of the invention is to provide a media formulation having particular utility for the recombinant production of bone morphogenic proteins. It is a further object of the invention to provide methods for consistently and reproducibly producing such serum-free media having improved cell cultivation properties.
These and other objects and features of the invention will be apparent from the description, figures, and claims which follow.